Download An in vitro beating heart model for long

Cardiovascular Research (2009) 81, 253–259
doi:10.1093/cvr/cvn299
An in vitro beating heart model for long-term assessment
of experimental therapeutics
Walter Habeler, Séverine Pouillot, Alexandra Plancheron, Michel Pucéat, Marc Peschanski, and
Christelle Monville*
INSERM/UEVE UMR 861, I-STEM, AFM, 5 rue Henri Desbruères, Evry 91030 Cedex, France
Received 17 March 2008; revised 20 October 2008; accepted 24 October 2008; online publish-ahead-of-print 3 November 2008
Time for primary review: 28 days
KEYWORDS
Heart slice cultures;
Cell therapy;
Human embryonic stem cells
Aims Within the framework of studies aiming at regenerative medicine for cardiovascular disease, we
have developed an in vitro model to analyse human embryonic stem (ES) cell engraftment into the
myocardium.
Methods and results This model is based on organotypic rat ventricular slices maintained in culture at
the air–medium interface on semi-porous membranes. Survival and differentiation of human cardiomyocytes derived from ES cells were then assessed for several months. In addition, we observed that ventricular tissue slices not only exhibited normal histology, but also rhythmic contractions till the end of
the experiments (up to 3 months). Similar results were obtained using ventricular slices obtained from
two human foetuses at 8 and 9.5 weeks of age. Calcium transients were associated with the beating frequency, and the pattern was modulated in a dose-dependent manner by epinephrine.
Conclusion Our data suggest that the organotypic heart slice culture on semi-porous membranes is a
relevant in vitro heart model for long-term histological and physiological studies.
1. Introduction
Development of cell therapy products to substitute for cardiomyocytes lost due to cardiovascular diseases has been a
major field of research over the past 10 years. A variety of
cells have been contemplated, including skeletal myoblasts,1–6 mesenchymal stem cells from bone marrow,7–10
and most recently embryonic stem (ES) cell-derived cardiomyocytes11–13 or cardiac committed stem cells.14 However,
functional results have been controversial, calling for a reassessment of the mechanisms of action of the transplanted
cells. It has, in particular, been difficult to determine
whether a significant proportion of cells did engraft,
whether some cells differentiated into fully mature
cardiac myocytes, or whether such a differentiation
process led to functional integration into the recipient
heart.14,15
The precise answers to these questions have been difficult
to provide for technical reasons, as data were derived from
in vivo experiments, in the absence of in vitro models that
would allow long-term assessment of cells transplanted
into heart tissue.
Preliminary attempts at designing such a model have been
made16–18 on the basis of organotypic heart slice cultures
designed to evaluate the toxicity of xenobiotics.19 In their
* Corresponding author. Tel: þ33 169908528; fax: þ33 169908521.
E-mail address: [email protected]
conditions, however, thin slice preparations of cardiac
tissue could only be experimentally utilized for ,2 weeks
in culture due to poor viability.16 Another, more complex
model has been recently designed by decellularizing the
heart while preserving the extra-cellular matrix and
reseeding it with cardiac or endothelial cells.20
We have reconsidered this important issue within the framework of studies aiming at substitutive cell therapy to the
heart using cardiomyocytes derived from human ES cells. We
have sought long-term survival of heart tissue slices, with
good histological preservation, by applying to the heart
the techniques designed for the so-called ‘chronic’ brain
slices,21–23 in which we managed recently to assess
implanted cells for several months.24,25
2. Methods
All experiments were performed in strict accordance with the recommendations of the European Ethical Committee (EEC) (86/609
EEC), the French National Ethical Committee (87/848), and by the
US National Institutes of Health (NIH Publication No. 85-23,
revised 1996) for the care and use of laboratory animals. The
human heart tissue was obtained following elective abortion, with
specific parental consent for scientific use as an ancillary to an
ongoing clinical protocol aiming at foetal neural grafting in patients
with Huntington’s disease (‘MIG-HD’, ref. NCT00190450). This investigation conforms to the principles outlined in the Declaration of
Helsinki.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
For permissions please email: [email protected].
254
2.1 Organotypic ventricular slice cultures, set up,
and characterization
Cultures were prepared from ventricular sections of 3-day-old rats
(n ¼ 30, Charles River, France) and from 8- and 9.5-week-old
human foetuses. The hearts were removed and placed in phosphatebuffered saline (PBS), the atria were removed, and the ventricles
sagitally sliced at 1 mm thickness using a rodent heart matrix
(Harvard Apparatus, UK) (see supplementary material online,
Figure S1). The heart slices were immediately transferred to a
Millicell-CM 0.4 mm membrane (Millipore, France), and the insert
was placed into a well containing 1 mL of medium in 6-well plates.
The culture medium consisted of Dulbecco’s modified Eagle’s/F12
medium (DMEM/F12) supplemented with 20% knockout serum replacement (KSR), 1% non-essential amino acids, 2 mM L-GLUTAMINE, 0.1%
b-mercaptoethanol, and 0.1% penicillin/streptomycin (Invitrogen,
France). Heart slices were maintained for 30–80 days at 378C in a
humidified atmosphere containing 5% CO2. Medium was changed
three times a week. Slices were observed under an inverted microscope every other day.
Rate and rhythm of heart slice beating were characterized in parallel as well as the localization of the most apparent contractions.
Further functional characterization of the contractile function of
ventricular slices was carried out on 23 rat and five human heart
slices, at 30 days after plating, using a stereomicroscope and a
temperature-controlled stage set at 378C. During these sessions,
each slice was incubated with 10, 100 nM, then 1 mM epinephrine
(Sigma, France). Beating frequency was monitored 3 min after
b-adrenergic stimulation.
Additional analysis was performed on three rat heart slices at 30
days after plating using a confocal laser scanning microscope
LSM510 (Zeiss), following incubation with 5 mM fluo-4 AM (Invitrogen) for 20 min at 378C.
2.2 Intra-slice cell implantations
Human ES cells (SA01, Cellartis, Sweden) were maintained on mitomycin C-inactivated mouse embryonic fibroblast feeder cells (STO,
ATCC) in DMEM/F12 medium supplemented with 20% KSR, 1% nonessential amino acids, 2 mM L-glutamine, 0.1% b-mercaptoethanol,
and 4 ng/mL basic fibroblast growth factor (Invitrogen). Undifferentiated human ES cells were harvested and resuspended in the
medium used for organotypic slice culture at a density of
1 105 cells/mL. An aliquot of 0.1 mL of this cell suspension was
then injected into heart slice cultures, using a 0.5 mL Hamilton
syringe fixed to the arm of a micromanipulator. Penetration of
human ES cells into rat heart slices was quantified by counting
human cells every 25 mm layer from the slice surface. Five sections
of five different heart slices were analysed.
The contraction rate of heart slices was monitored daily using a
Zeiss-Lumar V12 stereomicroscope.
2.3 Gene expression
Total RNA was extracted from heart slice cultures using TRIZOL
Reagent (Invitrogen), according to manufacturer’s protocol. RNA
(1 mg) was reverse-transcribed using SuperScript II RNase
H-Reverse Transcriptase (Invitrogen). cDNA was used as a template
for gene expression analysis of human cardiac markers. Real-time
quantitative PCR was performed using a LightCycler with SYBR
Green I master (Roche Diagnostic, France).
The following primer pairs were used: human-Oct-4 forward:
50 -CTT-GCT-GCA-GAA-GTG-GGT-GGA-GGA-A-30 and reverse: 50 -CT
G-CAG-TGT-GGG-TTT-CGG-GCA-30 ; human-Nanog forward: 50 -CAA-A
GG-CAA-ACA-ACC-CAC-TT-30 and reverse: 50 -CT-GCT-GGA-GGC-T
GA-GGT-AT-30 ; human-Gata4 forward: 50 -TCC-CTC-TTC-CCT-CCTCAA-AT-30 and reverse: 50 -TCA-GCG-TGT-AAA-GGC-ATC-TG-30 ; humanmyocyte enhancer factor 2c (Mef2c) forward: 50 -CGC-ATG-A
GA-GCC-GGA-CAA-ACT-30 and reverse: 50 -TGG-CTG-GAC-ACT-GGG-AT
G-GAG-30 ; human-atrial myosin light chain (MLC2a) forward:
W. Habeler et al.
50 -CGG-GGA-ACA-TCG-ACT-ACA-AG-30 and reverse: 50 -TTT-CCA-ATTTTG-CAA-CAG-AGT-TT-30 ; human-b myosin heavy chain (b-MHC)
forward: 50 -GGC-CCA-GAT-TCT-TCA-GGA-TT-30 and reverse: 50 -T
GG-CTG-GAC-ACT-GGG-ATG-GAG-30 ; human-atrial natriuretic peptide
(ANP) forward: 50 -TGT-TGC-CAT-GGA-GTT-GTG-AT-30 and reverse:
50 -GAG-AGG-CGA-GGA-AGT-CAC-C-30 ; human ubiquitin C (UbC)
forward: 50 -ATT-TGG-GTC-GCG-GTT-CTT-G-30 and reverse: 50 -TG
C-CTT-GAC-ATT-CTC-GAT-GGT-30 ; and rat ubiquitin C (UbC) was used
as an internal control forward: 50 -TCG-TAC-CTT-TCT-CAC-CAC-AGT-AT
C-TAG-30 and reverse: 50 -GAA-AAC-TAA-GAC-ACC-TCC-CCA-TCA-30 .
Amplification PCR reaction was performed with an initial denaturation
step at 958C for 5 min, followed by 50 cycles of denaturation at 958C
for 15 s, annealing at 608C for 15 s, and extension at 728C for 15 s.
Quantification of gene expression was based on the DeltaCt method
and normalized on human UbC. Melting curve analysis was used to
determine the specificity of PCR products, which was confirmed
using gel electrophoresis run.
2.4 Immunohistochemistry
Heart slices fixed in 4% paraformaldehyde for 2 h at 48C were cut
into 10 mm thick sections. The cryosections were washed with PBS
and then incubated for 1 h at room temperature with a saturating
solution consisting of 5% normal goat serum, 5% normal horse
serum in PBS 0.6% Triton X-100 (Sigma). Slides were incubated overnight at 48C with the following primary antibodies used at 1:400:
anti-human nuclei mouse monoclonal (HNA, Chemicon, France),
anti-connexin 43 mouse monoclonal (CX43, Chemicon), anti-rat
CD31 mouse monoclonal (BD Pharmigen, France), anti-troponin I
rabbit polyclonal (cTNI, Chemicon), anti-sarcomeric actinin mouse
monoclonal (Sigma), anti-desmin mouse monoclonal (Chemicon),
anti-human alpha-actinin mouse monoclonal (Chemicon), and
anti-Ki67 mouse monoclonal (Chemicon). After three washes with
PBS, the slides were incubated with a 1:500 dilution of fluorescentconjugated secondary antibodies for 1 h at room temperature. Secondary antibodies used were: Alexa-Fluor 488 goat anti-rabbit IgG
and Alexa-Fluor 555 goat anti-mouse IgG (Molecular Probes,
France). The specificity of all secondary antibodies was tested by
incubation of only the secondary antibody without a pre-primary
antibody incubation. All secondary antibodies were negative for
non-specific staining in our immunostaining condition. After three
washes, the slices were incubated with 1/10000 DAPI and then the
slides were mounted by using Fluoromount-G (CliniSciences,
France). The slides were observed under an epifluorescence microscope (Zeiss Imager Z1), and images were processed using the Axiovision software.
2.5 Statistical analyses
Statistical analysis was performed using analysis of variance
(ANOVA) and Student’s t-test. For time course of cell differentiation, statistical analysis was performed using ANOVA and
Dunnett’s test.
A value of P 0.05 was considered statistically significant.
3. Results
Macroscopic analysis of the organotypic rat heart slices
plated at the air–medium interface on semi-porous membranes demonstrated good overall preservation over time
(Figure 1A). Immuno-labelling of cardiomyocytes using troponin I and sarcomeric alpha-actinin antibodies confirmed
the regular organization of the cells with little, if any,
tissue disruption (see supplementary material online,
Figures S1B and S2). Connexin 43 immunolabelling also
revealed a dotted membranous distribution (Figure 1C), in
keeping with that observed in histological sections of the
heart. CD31 immunostaining additionally demonstrated the
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Organotypic rat heart slice cultures
In addition to human ES-derived cardiomyocytes and
intermingled with them, anti-Ki67 immunolabelling—
co-registering with HNA—revealed a significant proportion
of human cells that were still proliferating up to 2 months
after implantation (Figure 2E).
In contrast, immunostaining with endo- and ectodermal
markers was negative. There was neither teratoma in
heart slices transplanted with human ES cells.
3.2 Heart slices in culture show spontaneous
rhythmic contractions over months
Figure 1 Histology of rat heart slices after 1 month of culture (A) stereomicroscopic view, (B) anti-troponin I (green), (C ) anti-connexin 43 (green), and
(D) anti-CD31 (red) immunostaining. Nuclei are counterstained with DAPI
(blue).
presence of a regular network of blood capillaries in the
organotypic heart slices, comparable with that usually
observed in sections of the organ (Figure 1D).
3.1 Human embryonic stem cells differentiate into
cardiomyocytes following intra-slice transplantation
Sixty days after transplantation of undifferentiated human
ES cells into organotypic rat heart slices, HNA-positive
human cells were readily observed (Figure 2B). These cells
often clustered in several layers at the surface of the
slices, but also dispersed in the tissue itself, intermingling
with rat cells or accumulating around blood vessels. The
large majority of human cells (81 + 2.3%) was located
within 250 mm from heart slice surface (see supplementary
material online, Figure S3). Microscopic observation of the
cardiac matrix appeared intact, which may have prevented
the human ES cells from dispersing completely throughout
the tissue.
The human cells expressed markers of differentiated cardiomyocytes, including troponin I (Figure 2B) and humanspecific desmin (Figure 2C) and alpha-actinin (Figure 2D),
for both, of which no cross-reactivity was observed in the
surrounding rat tissue.
The expression of human specific genes was then monitored over time at 10, 20, 30, 40, 50, and 60 days after
injection of undifferentiated human ES cells into rat heart
slices using real-time PCR (Figure 3). We observed a downregulation of the undifferentiated stage markers Oct-4 and
Nanog over the first weeks of culture (Figure 3A). Starting
after 10 days and peaking at 1 month, the gene expression
of Gata4 and Mef2c increased, indicating cardiac mesoderm
differentiation (Figure 3B). After 2 months, cells expressed
MLC2a, b-MHC and ANP, and three cardiac markers
(Figure 3C).
Repeated stereomicroscopic observation to check preservation of tissue slices revealed that heart slices exhibited
spontaneous and regular rhythmic contractions during the
entire duration of the experiments (see supplementary
material online, Videos 1–3). This phenomenon happened
whether slices had received cell grafts or not and was thus
considered an intrinsic functional property of the heart
slice itself in our technical set up.
Contractions were most often visible macroscopically as
rhythmic alteration of one or several portions of the slice.
More rarely, the entire piece of tissue would rise partially
from the stage during contraction (see supplementary
material online, e.g. Video 3). The areas where contractions
were observed macroscopically also varied from one observation session to another.
Each contraction lasted less than a few 100 ms. The contraction rates were regular within the portion of the slice
over time during an observation session, whereas two
areas of the same slice could beat asynchronously. Beating
rates were low, below 60 bpm, and in general between 30
and 40 bpm. The monitoring of spontaneous beating frequency of an organtotypic rat heart slice at regular intervals
over 81 days showed very little contraction rate change over
time. Beating rate was usually recorded at 378C, but a
decrease in temperature down to 258C had no visible
impact on the frequency of contraction.
3.3 Rhythmic contractions of organotypic heart
slices on semi-porous membranes are associated
with calcium waves and modulated by epinephrine
Physiological mechanisms underlying heart slice beating
were explored by loading organotypic cultures with Fluo-4
AM ester. A confocal analysis of the temporal changes in fluorescence intensity showed rhythmic calcium transients in
different regions of the heart slices (see supplementary
material online, Video 2). Quantification of these calcium
oscillations showed that their rhythm was similar to that
of the contractions observed macroscopically. Calcium
waves were regular, not only over time but also in amplitudes at each local spot of analysis, where cells were
excited in synchrony (Figure 4A).
Cardiomyocytes in the organotypic heart slices also
responded to pharmacological manipulation, as demonstrated by applying epinephrine to the culture medium.
This treatment increased spontaneous beating frequencies
in a dose-dependent manner from 10 nM to 1 mM
(Figure 4B). In parallel, the confocal analysis of calcium
currents in heart slices loaded with fluo-4 AM showed a
dose-dependent increase in Ca2þ transient amplitude and
frequency with epinephrine (Figure 4C).
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W. Habeler et al.
Figure 2 Engraftment of human embryonic stem (hES) cells into organotypic rat heart slices. (A) Schematic diagram showing the geometry of the heart slice,
membrane, human cells grafted, and the cutting plane. The cryostat sections were perpendicular to the semi-porous membrane. (B) hES cells in the cardiac
parenchyma, 60 days after implantation. Human nuclei are specifically stained by anti-HNA (in red), cTNI immunostaining (in green) and DAPI staining
showing both human and rat cell nuclei (in blue), (C) anti-human desmin (red), (D) anti-human actinin (red), and (E) anti-human Ki67 (red) staining.
3.4 Organotypic human heart slice cultures at
air–medium interface on semi-porous membranes
One millimetre thick organotypic heart ventricular slices
were obtained from two human foetuses at 8 and 9.5
weeks of gestational age and plated at air–medium interface on semi-porous membranes. In keeping with the
results obtained with rat tissue, human organotypic heart
slices exhibited preserved histological markers of cardiomyocytes and blood capillaries. In parallel, regular rhythmic
contractions were observed up to the end of the observation
period (60 days) at a beating rate comparable with that of
rat slices (between 30 and 40 bpm, see supplementary
material online, Video 3). The addition of epinephrine to
the culture medium at 10, 100 nM, and 1 mM also increased
spontaneous beating frequency of human heart slices in a
dose-dependent manner (Figure 4D).
4. Discussion
The main outcome of this study is the set up of an organotypic
heart tissue culture method that allows long-term histological
and physiological studies, including the human. This result was
obtained by adapting to the heart the technique that allowed
chronic organotypic brain slices by maintaining them at the
air/medium interface on semi-porous membranes. The organotypic heart slice system is a useful substrate to analyse
after implantation long-term cardiomyocyte differentiation
of human ES cells. Preservation of cell excitability and
intercellular connections was associated with long-term
persistency of regular rhythmic contractions and calcium transients. This continuous physiological activity makes organotypic heart slices on semi-porous membranes suitable, in
addition, for pharmacological studies.
Attempts at setting-up organotypic heart slice cultures
have been made in the recent past by several authors, in a
search similar to ours for models allowing testing of experimental therapeutics.16–19 Standard histological staining in
combination with immunohistological analysis for cardiac
markers demonstrated that, originally, slices were structurally intact. The function of ion channels and receptors
could be preserved, in contrast to the observation made
on dissociated cardiomyocytes in culture. The main limitation in previous models was the survival time of the
tissue, which only allowed for short-term experimental
evaluation (,2 weeks). Physiological recordings were even
less encouraging, as they revealed a complete loss of
network activity within 3–4 days. Comparison of these
Organotypic rat heart slice cultures
257
Figure 3 Gene expression analysis of human cardiac markers. (A) Real-time polymerase chain reaction for Oct-4 and Nanog, (B) Gata4 and Mef2c, (C ) myosin
light chain 2a, b-myosin heavy chain, and atrial natriuretic peptide at different time points after implantation of undifferentiated human embryonic stem cells
into rat heart slices. *P 0.05 and ***P 0.001 when compared with human embryonic stem cells at day 0 (Dunnett’s test after analysis of variance). Data are
mean+SEM.
data with the vast literature concerning the much more
studied organotypic brain slice cultures led us to surmise
that, as was the case for the nervous system, the poor preservation of heart tissue over time in those studies was
related to the immersion of the slice in the culture
medium.26 Long-term preservation of organotypic brain
slices has been readily obtained by replacing immersion by
techniques that allowed direct oxygenation of the slices,
either using slow rotation to create continuous changing of
the liquid–gas interface,27 or by placing slices at the air–
medium interface on a semi-porous membrane.22 We chose
to develop the latter technique because it was suitable for
subsequent intra-slice cell implantation.24,25 Our results
confirmed the long-term preservation of the organotypic
slices also for the heart in those air–medium interface conditions. It is interesting to underline the fact that this longterm culture system preserved not only individual cells, but
even more the pluricellular structures they form in the
tissue. This was exemplified by the overall organization of
well-aligned cardiomyocytes potentially linked by gap junctions, the preservation of membrane-bound connexins, the
presence of a dense network of blood vessels—although
without blood—and the demonstration of calcium waves
spreading along local arrays of excitable cardiomyocytes.
However, Cx43 was localized not only at intercellular contacts but more widespread in the cells, possibly a consequence of injury at the time of slice preparation.28
The original goal of the set-up of chronic heart slices was
the search for an in vitro heart substrate to analyse grafted
cells. The rat heart slices allowed us to address this issue
quite successfully, as human ES cells implanted at a fully
undifferentiated stage not only survived and integrated
with the rat tissue but also readily differentiated into
cardiomyocytes. This latter result is of particular interest
as it was obtained without any priming of the cells to be
implanted with cytokines that push ES cells towards the
cardiomyogenic lineage. Embryoid bodies generated from
stem cells primed with TGF-beta, and BMP2 demonstrated
an increased potential for cardiac differentiation, in
vitro.29–30 In vivo, transplantation of stem cells into heart
also resulted in cardiac differentiation, provided that
TGF-beta/BMP2 signalling was intact,15,29 but this capacity
remained limited unless cardiomyocytes had been
previously damaged.10,31 Organotypic heart slices seem,
therefore, to replicate the latter situation as they provide
a similarly appropriate environment for cardiomyogenic
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W. Habeler et al.
Figure 4 Characterization of spontaneous contraction of rat and human heart slices. (A) Ca2þ spiking within selected ROI of a heart slice loaded with Fluo-4.
Recordings are expressed as DF/F0, where F0 is the lowest level of fluorescence. (B) Functional effects of epinephrine on heart slices in organotypic cultures.
Dose–response increase in frequency of rat slice beating. **P 0.01 and ***P 0.001 when compared with untreated controls (Student’s t-test after analysis of
variance). Data are mean+SEM, n ¼ 23. (C) Ca2þ spiking recorded after loading with Fluo-4 in rat heart slice cultures at rest and during application of 1 mM
epinephrine. (D) Increase in frequency of human heart slice beating during epinephrine application vs. control. Mean+SEM, n ¼ 5.
259
Organotypic rat heart slice cultures
differentiation, indicating the preservation of the cardiac
paracrine pathway required for therapeutic benefit of
stem cell transplantation in the diseased heart.
As an unexpected outcome of this study, preservation of
spontaneously functional excitable networks was maintained for several months in the heart slices. The rhythm
of the contractions was slow, in keeping with extranodal
pacemakers in the ventricular slices. Notwithstanding that
limitation, the slice model appears as a promising substrate
for physiological analysis. It indeed displays regular contractions associated with calcium waves that spread over networks of cardiomyocytes that respond readily to the
activation of b-adrenergic receptors. This will allow for
functional studies looking for the effects of drugs on cardiomyocytes, whether for pharmacological or for pharmacotoxicological purposes.
10.
11.
12.
13.
14.
15.
Supplementary material
Supplementary material is available at Cardiovascular
Research online.
16.
17.
Acknowledgements
We thank Xavier Nissan for assistance with real-time PCR analysis
and Marc Lechuga with statistical analysis.
18.
Conflict of interest: none declared.
19.
Funding
20.
This study has been supported in part by additional grants from the
Agence Nationale de la Recherche (CSCelo) and the cluster Medicen
Paris Region (IngeCELL).
21.
22.
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